Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Dopaminergic modulation of motor neuron activity and neuromuscular function in Drosophila melanogaster
Introduction
Neuromodulators are known to function as important signaling molecules in animals, and can alter activity of the central and peripheral nervous systems [25], [42], [43]. One neuromodulator in particular, serotonin (5-HT), is involved in the behavioral expression of dominance and aggression in widely evolutionarily diverged species; the role of 5-HT in aggression has been explored in crustaceans [31], [45], [51] and humans [10], [11], [30], [50]. In Drosophila melanogaster, 5-HT modulates heartbeat [26], [33], and voltage dependence of delayed rectifier and Shaker potassium channels [24]. Dopamine and 5-HT have been identified in fly heads, which suggests that neural activity is regulated by these compounds.
Little is known about the neuromodulatory roles of dopamine. It has been shown to act as a neurotransmitter in Drosophila, modulating female sexual receptivity and habituation, a form of learning [36], [37]; however, it is also required for the normal development of both gonadal and other tissues [35]. We propose dopamine plays a functional role in behavioral modulation, neuroendocrine activity, and in development. Functional roles for dopamine in a variety of insects is well substantiated [21]; for example, the dopamine receptor densities in the brain of the honey bee are altered during development [29] and in relation to certain behaviors in bees [49]. The cloning and functional characterization of dopamine [22] and octopamine [23] receptors from Drosophila have been described. Localization and characterization of these receptors at the NMJ will hopefully be forthcoming.
To understand the neuromodulator effects of 5-HT and dopamine in D. melanogaster, we examined the synaptic efficacy of D. melanogaster motor neurons at the neuromuscular junctions of high- and low-output terminals during the third instar larva stage by recording evoked and spontaneous quantal currents for quantal analysis. With this approach, one can assess pre- as well as post-synaptic differences induced by the addition of particular neuromodulators. Our results suggest a functional role for dopamine as a neuromodulator at the neuromuscular junction.
Section snippets
Chemicals
Serotonin hydrochloride (5-HT), dopamine hydrochloride and physiological salts were obtained from Sigma. The 5-HT or dopamine solution was made the day of experimentation. Anti-serotonin antibody was purchased from Incstar (ICN) and secondary antibodies (anti-rat IgG and anti-rabbit IgG, conjugated to fluorescein) were purchased from ICN.
Animals and dissection
The wild-type Canton S fly strain was raised at 19–20°C on standard cornmeal-agar-dextrose-yeast medium. Only wandering third instar larvae were used for the
HPLC analysis
Quantification of dopamine and 5-HT in whole larvae revealed different levels of expression. Systemic larval dopamine levels decrease from approximately 0.7 μg/ml in second instar to less than 0.4 μg/ml in the mid-third instar (larvae were homogenized in 3 μg/ml of perchloric acid, see Section 2, Fig. 1A). However, tyrosine hydroxylase levels are known to peak at the hatching/first instar and late third instar/pupariation boundaries [39]. Systemic 5-HT levels, although substantially lower than
Discussion
In the fruit fly, dopamine is known to have an effect on behavior as well as in development. If dopamine reserves are depleted in the larva, they will become aphagic and succumb to death [35]. In adults, depletion of dopamine alters female sexual receptivity and a learning behavior [36], [37]. If the depleted dopamine levels are restored by L-DOPA, the previously altered behaviors are rescued. HPLC quantification of dopamine and 5-HT in whole larvae revealed distinct levels of expression which
Acknowledgements
Illustrations were provided by the courtesy of Hye Won Cooper. We thank Joe Wegrzyn (University of Kentucky) for editorial assistance. Funding was provided by University of Kentucky Research and Graduate Studies Office (R.L.C.) and NSF grants IBN-9808631 (R. Cooper) and IBN-9423616 (W. Neckameyer).
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